Lactic Acid Bacteria and Formic Acid Improve Fermentation Quality and Beneficial Predicted Functional Characteristics in Mixed Silage Consisting of Alfalfa and Perennial Ryegrass

The purpose of the present study was to investigate the effect of additives on the fermentation properties of ensiled mixed alfalfa and perennial ryegrass silage in the karst terrain of Southwest China. A mixture of alfalfa and perennial ryegrass was ensiled at a ratio of 3:7 using three experimental treatments: (1) CK (without additives) and distilled water (5 mL kg⁻¹ fresh weight (FW)); (2) FA and formic acid (88%) (5 mL kg⁻¹ FW); and (3) LAB combined with the application of <i>Lactiplantibacillus plantarum</i> and <i>Lentilactobacillus buchneri</i> (2 × 10⁷ cfu/g FW). All samples were packed manually into polyethylene bags, and three polyethylene bags from each treatment were sampled on days 7, 15, and 45. The findings demonstrated that the pH values of all the mixed silages gradually decreased during ensiling. The lactic acid (LA) and acetic acid (AA) contents increased gradually with ensiling time and peaked after 45 days of ensiling. After 45 days of ensiling, the FA and LAB groups effectively preserved the nutrient content of the mixed silage, which presented a reduced neutral detergent fiber and acid detergent fiber content (<i>p</i> < 0.05) and higher water-soluble carbohydrate content (<i>p</i> < 0.05) than the CK group. The fermentation quality of the mixed silages in the FA and LAB groups improved, as indicated by higher (<i>p</i> < 0.05) LA contents and lower (<i>p <</i> 0.05) pH and ammoniacal nitrogen contents after 45 days of ensiling compared to those in the CK group. As fermentation progressed, the abundance of harmful microorganisms (<i>Hafnia obesumbacterium</i>, <i>Enterobacteriaceae</i>, and <i>Sphingomonas</i>) and beneficial microorganisms (<i>Lactiplantibacillus</i> and <i>Lentilactobacillus</i>) decreased and increased, respectively. In addition, compared to those in the CK group, the FA group had higher abundances of “lipid metabolism” and “biosynthesis of antibiotics” and lower abundances of “membrane transport”. Briefly, the results of this study suggest that the incorporation of FA and LAB additives could improve the quality of fermented mixed silage, and that FA is better than LAB. This information is useful for combining forage resources to satisfy the requirements for high-protein feed and for manufacturing ruminant feed annually.